Here, we demonstrate that high-quality graphene nanosheets can be produced in large quantities by liquid-phase shear exfoliation under ambient conditions in organic solvents, such as 1-methyl-2-pyrrolidinone (NMP), with the assistance of urea as a stabilizer. We can achieve low-defect graphene (LDG) using this approach, which is relatively simple and easily available, thereby rendering it to be an efficient route for the mass production of graphene. We also demonstrate the electrochemical sensing of an LDG-modified electrode for the determination of doxorubicin (DOX). The sensor shows an enhanced electrocatalytic property towards DOX, leading to a high sensitivity (7.23 × 10 Graphene can be applied to everything from electronics and pharmaceuticals to sensing, biomedical and energy devices ,2. With To circumvent these issues, one of the most promising techniques is the physical exfoliation of layered graphite to graphene, which can lead to the mass production of low-defect graphene (LDG) . The mecIn this study, we demonstrated that graphite can be exfoliated to give large quantities of graphene nanosheets by the liquid-phase shear exfoliation method, using a homogenizer in NMP as an organic solvent, with the assistance of urea as a stabilizer, under ambient conditions. Remarkably, we could obtain non-oxydized LDG sheets on a large scale under optimal conditions. Furthermore, the LDG was used to modify an electrode for fabricating the electrochemical sensor of doxorubicin (DOX). This sensor showed a good sensitivity and selelctivity for the electrochemical detection of DOX. This approach has a great potential in the industrial-scale production of graphene with good quality for practical applications.The chemicals and materials were purchased from Sigma-Aldrich, Inc. , as follows: natural graphite powder, NMP, phosphate buffer saline , glucose, KCl, NaCl, urea, doxorubicin HCl, tryptophan and commercial graphene. The deionized (DI) water was prepared using water purification systems .™ workhead. The absorbance of the exfoliated LDG was measured by UV-vis spectroscopy . The concentrations of the LDG were calculated from the standard curve for the absorbance at 660 nm of the commercial graphene solution. The Raman spectroscopy was performed using an EnSpectr R532 Raman spectrometer . Transmission electron microscopy (TEM) was carried out on a JEOL 2010 device  with an accelerating voltage 200 keV. The X-Ray diffraction (XRD) patterns were recorded using a Rigaku SmartLab X-ray diffractometer  with Cu Kα source. Fourier transform infrared spectroscopy (FTIR) was performed with a NICOLET iS10 . The X-ray photoelectron spectroscopy (XPS) signals were recorded using a Thermo Scientific K-Alpha XPS system  equipped with a micro-focused monochromatic Al Kα X-ray source (1486.6 eV). The electrochemical measurements were performed with a CHI 660D electrochemical workstation . A conventional three-electrode cell was used with a Pt wire as a counter electrode, an Ag/AgCl electrode was used as a reference electrode and a bare glassy carbon electrode , or modified electrode, was used as a as working electrode at room temperature.The shear exfoliation was performed with an L5M-A mixer homogenizer  with a Square Hole High Shear ScreenThe LDG samples were prepared by the liquid-phase shear exfoliation method using a high-speed mixer homogenizer, as shown in 2O3 powder and washed in an ultrasonic bath for 10 min, then rinsed with DI water thoroughly. After that, 5 μL of 1 mg/mL LDG in ethanol was drop-casted onto the GCE and dried with an infrared spectroscopy (IR) lamp for a few minutes, then rinsed with DI water several times. Before the electrochemical sensing experiments, the LDG-modified GCE was activated electrochemically by cyclic voltammetry (CV) in 10 mM PBS buffer solution (pH 7.4); the potential was scanned from 0 to 1.0 V (vs. Ag/AgCl) three times with a 5 mV/s scan rate.Before the electrode fabrication, the GCE was polished with 1.0, 0.3, and 0.05 μm Al−1 (2D band) for both graphite and LDG. Using the ratio of peak intensities ID/IG in Raman spectra, the level of disorder in graphene can be characterized. As the disorder in the graphene increases, D/IGI displays two different behaviors, as follows: (i) at a low-defect concentration, like pure graphite materials, D/IG ≃ 1/Ld2I (Stage 1); (ii) at a relatively high defect concentration, like produced graphene by chemical or physical exfoliation, D/IG ≃ Ld2I (Stage 2), where dL represents the mean distance between two defects [D/IGI ≃ 3 [D/IGI value increased as the level of defects decreased. The exfoliation caused an increase in the ID/IG value to 0.39 in the LDG, which is higher than that of graphite (0.16), but considerably lower than that of GO reported previously [−1) when compared to the 2D band of graphite. A shoulder peak on G band, along with the D, G and 2D band, appeared at 1616 cm−1 in the LDG, which is namely D band and is evidence of few-layer graphene. These results successfully demonstrate the reduction in the number of graphene layers and the subsequent formation of few-layer graphene with a low density of defects [To confirm the production of the LDG sheets, as described in  defects ,23. The D/IG ≃ 3 . So, in eviously . This sheviously . In addi defects .D/IGI) using Raman spectroscopy . The effect of the shearing times in aqueous urea solution and NMP, respectively, were further evaluated in the range of 1 to 5 h  for the detection of DOX. DOX, as one of the clinically important anti-cancer drugs, has been extensively used in the treatment of various cancers. However, long-term clinical use of DOX is limited due to its side effects, such as systemic toxicity, drug resistance, and life-threatening heart damage. Therefore, the development of highly sensitive analytical techniques for DOX detection is of great significance in clinics and pharmaceutics.3Fe(CN)6. As shown in pΔE = 70 mV) of LDG-GCE is smaller than that of bare GCE (119 mV), while the peak currents for LDG-GCE are significantly higher, indicating that the LDG-GCE electrode exhibited a better electrochemical performance. The EIS experiments also showed better electron-transfer characteristics for LDG-GCE. The charge transfer resistance (ctR) of LDG-GCE was estimated to be 25.9 Ω from the diameter of a semicircle in the high frequency region of a Nyquist plot (ctR = 168.8 Ω). This suggests that rapid electron transfer occurred on the LDG-GCE surface.Before the electrochemical sensing experiments, CV and electrochemical impedance spectroscopy (EIS) were performed to investigate the charge transfer ability of LDG-GCE in 0.1 M KCl solution containing 3 mM of Kist plot B, which −1 μM/μA with LDG-GCE, which is higher than bare GCE (3.54 × 10−1 μM/μA). Moreover, the limit of detection (LOD) for LDG- GCE (39.3 nM) at an S/N ratio of 3 was also much better than that of bare GCE with LDG-GCE (254 nM) and bare GCE. These results suggest that the LDG-GCE exhibited an excellent performance with respect to the detection of DOX. To evaluate the practical applications in DOX analysis, chronoamperometric measurements were carried out in 0.1 M PBS (pH 7.4). i (nA) = 1.06 × 10−8 + 0.00828 C (μM) in S/N = 3). The electrochemical detection of DOX was performed with differential pulse voltammetry (DPV) and chronoamperometry in 10 mM PBS solution (pH = 7.4) containing various concentrations of DOX, using LDG-GCE. D/IG value from the Raman spectra. The characterization experiments confirmed that the graphene sheets, produced by shear exfoliation with optimized parameters, are single layered, or few-layered at least, with negligible defects. In addition, using the LDG as a modification material for an electrode, we have fabricated an LDG-GCE electrochemical sensor for DOX, demonstrating sensitive and selective DOX analysis. Therefore, the proposed approach is promising for the industrial production of graphene for numerous applications.We have demonstrated the scalable production of graphene sheets with high quality by a simple and facile method, via NMP-mediated shear exfoliation of graphite with the assistance of urea. The successful production of LDG on a large scale was possible using a high speed mixer homogenizer by optimizing the processing parameters for the shear exfoliation, such as shearing speed, urea concentration, and shearing time by comparing the produced graphene concentration and I


A method of combining the AlGaN/GaN superlattices and Mg delta doping was proposed to achieve a high conductivity p-type GaN layer. The experimental results provided the evidence that the novel doping technique achieves superior p-conductivity. The Hall-effect measurement indicated that the hole concentration was increased by 2.06 times while the sheet resistivity was reduced by 48%. The fabricated green-yellow light-emitting diodes using the achieved high conductivity p-type GaN layer showed an 8- and 10-times enhancement of light output power and external quantum efficiency, respectively. The subsequent numerical calculation was conducted by using an Advanced Physical Model of Semiconductor Device to reveal the mechanism of enhanced device performance. This new doping technique offers an attractive solution to the p-type doping problems in wide-bandgap GaN or AlGaN materials. A (~200 meV) at room temperature which is much larger than the donor  activation energy ED (~15 meV) in GaN [18 cm−3) epilayer is still difficult to achieve. Heavy Mg doping seems like the most effective way to achieve a high hole concentration of p-type GaN epilayer, but the hole mobility will decrease due to the serious impurity scattering. Furthermore, the increased dopant concentration will lead to a degradation of GaN epilayer crystalline quality. Various methods have been proposed to improve p-conductivity in GaN. Kozodoy et al. utilized the AlGaN/GaN superlattice (SLs) structure only doping in the interface to promote the Mg accepter ionization [xGax1−N layer [III-Nitride and related alloys are attractive materials due to their immense potential in optoelectronic applications including the ultraviolet and visible range ,6,7,8,9.) in GaN . Therefo) in GaN ,12, but nization . HoweverxN layer . HoweverxN layer ,15,16,170.05Ga0.95N electron blocking layer (EBL), and Mg doped p-type layer. The p-type layer for sample A was uniform Mg doped GaN layer which was used as reference sample. Triethylgallium, trimethylaluminium, and ammonia were used as source compounds. Hydrogen gas was the carrier gas. Bis-cyclopentadienyl-magnesium (CP2Mg) was used as a Mg dopant source and the CP2Mg flow was kept at 1500 sccm. For sample B, the p-type layer was divided into three parts. The top and bottom parts were uniform Mg doping GaN layer. A 9-periods p-type 4-nm-Al0.05Ga0.95N/3-nm-GaN structure was adopted in the middle part of sample B. In order to achieve delta doping at the SLs structure in sample B, the flow of CP2Mg was constant throughout the entire p-type layer growth, while the growth process had a 6-s pause at each SL interface and the Mg source was still opened. This growth process leads to an intentional increased concentration of Mg dopants at the SLs interfaces and actually implements a Mg delta doping in the middle part of sample B. To further verify the improvement of device performance by novel doping technique, two green-yellow light emitting diodes (LED) are fabricated using the two different p-type GaN layer. 0.05Ga0.95N/GaN SLs are employed to regulate the stress. The InGaN/GaN MQWs consist of ten 2 nm In0.45Ga0.64N quantum wells embeds in eleven 15 nm GaN quantum barriers. The In0.45Ga0.64N/GaN MQWs are terminated by an Al0.05Ga0.95N EBL. The top of the LED structure is the Mg doped p-type layer. The LED A adopts the uniform Mg doped p-GaN layer while LED B adopts the p-type layer structure of sample B above. After the growth, standard chip processing is carried out and the size of the LED chip is 300 μm × 325 μm. In order to reveal the mechanism of enhanced p-conductivity and device performance, an Advanced Physical Model of Semiconductor Device (APSYS) simulation is adopted based on the structure above. The main parameters set in the simulation are the polarization shielding coefficient of 0.5, the auger composite coefficient of 1 × 10−43/(m6·s), the band offset ratio of 0.7 and the carrier lifetime of 10 ns.In order to investigate the influence of novel doping method on the p-conductivity of GaN film, two p-GaN structures were grown by AIXTRON CRUIS I metal-organic chemical vapor deposition (MOCVD) system on the c-plane patterned sapphire substrates (PSS). 18 cm−3, which is 2.06 times higher than that of sample A, and it is an order of magnitude higher than previous reported hole concentration of p-GaN grown by MOCVD [To characterize the different conductivities of the two p-GaN samples, the Hall-effect measurement was performed at room temperature using a Lake Shore 8400 Series at a magnetic field of 0.5 T. The measured hole concentrations, hole mobilities, and sheet resistivities are shown in by MOCVD ,20. At tc-face sapphire substrate with wavelength of 559 nm, the LOP was 11 mW at an injection current of 20 mA [c-plane sapphire substrate with a size of 300 × 300 μm2. But the dominant wavelength and output power at 20 mA were 556.3 nm and 0.24 mW, respectively [In order to verify the effectiveness of p-conductivity on the performance of LED, of 20 mA . Howeverectively . CompareIn order to clarify the mechanism of novel doping methods enhanced the p-conductivity of GaN film, the calculated hole concentrations distributions and energy band diagrams of sample B are given in In addition, for take a deep insight into the improved p-conductivity on the performance of LED, the APSYS simulated results of LED performance is presented in In summary, we proposed a method of combining the AlGaN/GaN SLs and Mg delta doping to achieve a high conductivity p-type GaN. Hall-effect measurement showed the hole concentration of sample B was increased by 2.06 times while the sheet resistivity was reduced by 48% compared to sample A. In order to verify the enhancement of the GaN based photoelectric device performance by improved p-conductivity, a green-yellow LED was fabricated using the optimized high conductivity p-GaN layer and the EQE was increased by 10 times. The APSYS simulated results illustrated the enhancement mechanism of p-conductivity and LED performance. The novel technique of p-type doping in this work are very promising for the achievement of high-efficiency GaN based LEDs and electronic devices.


MicroRNAs (miRNAs) play a vital role in the development of ovarian cancer (OC). The aim of this study to investigate the prognostic value and potential signaling pathways of hsa-miR-9-5p (miR-9) in OC through literature review and bioinformatics methods.The expression of miR-9 in OC was assessed using the public datasets from the Gene Expression Omnibus (GEO) database. And a literature review was also performed to investigate the correlation between miR-9 expression and the OC prognosis. Two mRNA datasets (GSE18520 and GSE36668) of OC tissues and normal ovarian tissues (NOTs) were downloaded from GEO to identify the differentially expressed genes (DEGs). The target genes of hsa-miR-9-5p (TG-miR-9-5p) were predicted using miRWALK3.0 and TargetScan. Then the gene overlaps between DEGs in OC and the predicted TG-miR-9-5p were confirmed using a Venn diagram. After that, overlapping genes were subjected to Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, a protein-protein interaction (PPI) network was constructed using STRING and Cytoscape, and the impact of hub genes on OC prognosis was analyzed.P < 0.05), PRRX1 , SMC2 , SMC4 , and VCAN .It was found that OC patients with miR-9 low expression had poor prognosis. A total of 107 DEGs related to both OC and miR-9 were identified. Dozens of DEGs were enriched in developmental process, extracellular matrix structural constituent, cell junction, axon guidance. In the PPI network analysis, 5 of the top 10 hub genes was significantly associated with decreased overall survival of OC patients, namely FBN1 (HR = 1.64, Low expression of miR-9 indicates poor prognosis of OC patients. MiR-9 plays a crucial role in the biological process of OC by binding to target genes, thus affecting the prognosis of patients. Ovarian cancer (OC) is one of the most common gynecological malignancies, which poses a serious threat to women’s life and health. OC is clinically characterized by insidious- and late-onset, high grade of malignancy, high metastasis rate, and poor prognosis. Its incidence is increasing yearly, and its mortality rate ranks first compared with other female genital tract malignancies, such as endometrial cancer, cervical cancer and vulvar cancer , 2. The Because of the low survival rate and high recurrence rate, it is of great significance to find new targets related to the prognosis of OC, and explore the specific mechanism, so as to perform personalized treatment and to improve clinical efficacy and quality of life of OC patients. Besides,, new targets will also have a significant impact on the detection of cancer recurrence and the guide to rehabilitation.MicroRNAs (miRNAs) are a class of highly conserved endogenous non-coding small RNAs composed of 18–22 nucleotides , 7, whicNumerous studies showed that there was a significant correlation between miR-9 expression and the prognosis of various cancers, such as breast cancer , osteosahttp://www.ncbi.nlm.nih.gov/geo/). The search strategy was as follows: (“ovarian cancer” OR “ovarian carcinoma” OR “ovarian tumor”) AND . The datasets related to miR-9 expression in OC tissues and normal ovarian tissues (NOTs) were included in our analysis.To obtain gene chip profiles of OC, a search was performed in the Gene Expression Omnibus database . Based on the groups , the data in the original files were sorted into a Excel table. By using the gene probe file provided by the platform, the gene probes in the sorted Excel document were converted into the gene symbols. Finally, the gene expression data of miR-9 in OC and NOTs were screened.PubMed, Web of Science, Cochrane Library, and Embase databases were comprehensively searched for the literature on the correlation between miR-9 expression and prognosis of OC. The time of literature retrieval was up to November 2020. The search strategy was as follows: (“microRNA-9” OR “miRNA-9” OR “miR-9”) AND (“ovarian cancer” OR “ovarian carcinoma” OR “ovarian tumor”). Literature retrieval was performed independently by two investigators and finally cross-checked.The studies published in English on the relationship between miR-9 expression and OC prognosis were included. While reviews, nonclinical studies, case reports, abstracts were excluded. The quality evaluation of the included studies was performed according to the Newcastle-Ottawa scale (NOS) . Only thGene expression profiles of datasets GSE18520 and GSE36668 were obtained from GEO. The dataset GSE18520 consists of 53 OC samples and 10 NOTs samples, while GSE36668 consists of 4 OC samples and 4 NOTs samples. The data were analyzed on the GPL570 platform Affymetrix Human Genome U133 Plus 2.0 Array .P-value  was calculated using Benjamini-Hochberg method for controlling the false discovery rate (FDR), thus correcting false positives. Cut-off criterion was defined as P < 0.05 and |log2 (FC)| > 1. The platform annotation files downloaded from the database were adopted to convert the probe data in the matrix files into gene symbols.To identify differentially expressed genes (DEGs) between OC tissues and NOTs, we employed the Limma package (version 3.6.3) in R/BioManager for processing. The adjusted http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/miRretsys-self.html) and TargetScan (http://www.targetscan.org/vert_72/) were used to predict the target genes of hsa-miR-9-5p (TG-miR-9-5p). The overlapping genes between DEGs in OC and TG-miRNA-9-5p were revealed, and TG-miR-9-5p was also predicted using the online tool Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/). The gene overlaps were analyzed by Gene Ontology (GO) and visualized using the Bingo plugin of Cytoscape software (version3.7.2). Kyoto Encyclopedia of Genes and Genomes (KEGG) was analyzed by using DAVID database. FDR < 0.05 was considered as the cutoff criterion. In addition, the protein-protein interaction (PPI) network based on the gene overlaps was constructed by using STRING 11.0 (https://string-db.org/), and the interactions were visualized with CytoHubba plugin for Cytoscape software (version 3.7.2). Then the hub genes of PPI network were obtained. A confidence score of C ≥ 0.7 was set as the cutoff criterion.The online websites miRWALK3.0 (www.kmplot.com). The Kaplan-Meier plotter is capable to assess the effect of 54 k genes  on survival in 21 cancer types, including breast (n = 6234), ovarian (n = 2190), lung (n = 3452), and gastric (n = 1440) cancers. Sources for the databases include GEO, European Genome-phenome Archive (EGA), and The Cancer Genome Atlas (TCGA). OC patients were divided into the high expression group and the low expression group based on the median expression of hub genes. The overall survival risk of OC was evaluated using Kaplan-Meier method, and the hazard ratio (HR) was used as a measure of effect.The effect of the top 10 hub genes obtained in the PPI network on OC prognosis was assessed using Kaplan-Meier plotter . Independent two-sample t-test was used for analysis. Pooled analysis of miR-9 expression levels in different GEO datasets was performed using a random-effects model. If HR and its 95% CI of miR-9 expression and OC survival risk were provided in the included literature, they were directly adopted. If HR was not directly provided in the literature, but with survival curves, the data were then extracted using Engauge Digitizer software and then HR was calculated. Overall survival (OS) and progression-free survival (PFS) were used to evaluate the effect of miR-9 expression on OC prognosis. A fixed-effects model was used for the pooled analysis of these prognostic indicators. P < 0.05). And no significant difference was identified in the GSE23338 dataset. The above comparison results are shown in Table P = 0.952). The forest plot is shown in Fig. The screening process of GEO datasets is shown in Fig. The screening process of the literature is shown in Fig. In the GSE18520 and GSE36668 datasets, 3146 and 2051 DEGs were identified, respectively. Volcano plots of gene expression differences in these two datasets are shown in Fig. As shown in Table The PPI network of miR-9-related DEGs was constructed with 101 nodes and 48 edges, which included 50 up-regulated genes and 51 down-regulated genes , PRRX1 , SMC2 , SMC4 , and VCAN . The differences were statistically significant.The prognostic values of the top 10 hub genes selected from the PPI network were evaluated using Kaplan-Meier plotter. The results showed that 5 of the top 10 hub genes were significantly associated with the risk of survival in OC patients. High expression of these 5 hub genes led to a decrease of survival rate in OC patients , Vestigial like family member3 (VGLL3). CALB2 is a 29-kDa calcium-binding protein of the EF-Hand family, which is a family of proteins containing calcium-binding motifs composed of two helices (E and F) . CALB2 hOur study also found 5 hub genes with a significant correlation with OC prognosis, such as FBN1, PRRX1, and SMC4. Their high expression contributes to the poor prognosis of OC patients. FBN1 is the coding gene for fibrillin-1 and the main component of extracellular matrix microfibers, which is crucial in maintaining the morphological integrity and normal function of connective tissue . StudiesThe findings of this study have great and far-reaching significance for the clinical targeted therapy of OC patients. It is a long-term and arduous task for medical workers to explore the mechanism of miRNA in OC, to find effective targets, to develop effective targeted drugs for targeted therapy, so as to prolong the survival time of OC patients and improve their quality of life.In summary, miR-9 is critical in the biological processes of OC. However, further in vitro and in vivo experiments are still required for its pathogenesis, thus validating the role of miR-9-regulated molecular networks in OC.


In addition, in vitro cytotoxicity and cellular uptake of DOX-HSA-MPs were evaluated using the lung carcinoma cell line A549. The results demonstrated that DOX-HSA-MPs reduced the cell metabolic activities after 72 h. Interestingly, DOX-HSA-MPs were taken up by A549 cells up to 98% and localized in the cell lysosomal compartment. This study suggests that DOX-HSA-MPs which was fabricated by CCD technique is seen as a promising biopolymer particle as well as a viable alternative for drug delivery application to use for cancer therapy.Doxorubicin (DOX) is an effective anthracycline antibiotic drug which is commonly used in a broad range cancer therapy. However, due to dose depending side effects and toxicity to non-cancerous tissues, its clinical applications are restricted. To overcome these limitations, human serum albumin (HSA) has been investigated as a biocompatible drug delivery vehicle. In this study, human serum albumin submicron particles (HSA-MPs) were fabricated by using the Co-precipitation–Crosslinking–Dissolution technique (CCD technique) and DOX was loaded into the protein particles by absorption. DOX-HSA-MPs showed uniform peanut-like shape, submicron size and negative zeta-potential (−13 mV). The DOX entrapment efficiency was 25% of the initial amount. The in vitro release in phosphate buffered saline pH 7.4 was less than 1% within 5 h. In contrast, up to 40% of the entrapped DOX was released in presence of a protein digesting enzyme mixture (Pronase Streptomyces peucetius var. caesius in the 1970s [®, Lipodox®) and unpegylated (Myocet®) (FDA-approved) [Doxorubicin (DOX) was first extracted from he 1970s ,3. Howevhe 1970s  and its he 1970s ,6 and skpproved) , encapsupproved)  and Dox-pproved)  are beinpproved) .In recent years, albumin has been actively explored and considered as a reliable and efficient carrier for drug delivery systems ,14,15. AIn our group a successful technique, known as Co-precipitation–Crosslinking–Dissolution (CCD) technique was developed, which was used to fabricate biopolymer particles ,23,24. TIn the present study, we propose a procedure to fabricate doxorubicin loaded albumin microparticles (DOX-HSA-MPs) by a slightly modified CCD-Technique, which releases Doxorubicin only after endocytosis and digestion of the particles. The hypothesis was tested with pulmonary adenocarcinoma cells (A459 cell line) presenting albumin receptors and human bronchial epithelial cells (BEAS-2B cell line), without albumin receptors. The obtained DOX-HSA-MPs were characterized concerning size, morphology, surface charge as well as for their drug release profile, cellular uptake by A459 and BEAS-2B cells as well as for cytotoxicity to these cells.2·EDTA), glutaraldehyde (GA), magnesium chloride (MnCl2), sodium carbonate (Na2CO3), phosphate buffered saline (PBS), pH 7.4, fluorescein isothiocyanate (FITC), glycine and sodium borohydride (NaBH4) were purchased from Sigma-Aldrich . Sodium hydroxide (NaOH) was purchased from Carl Roth GmbH . Ampuwa and sterile 0.9% NaCl solution were purchased from Fresenius Kabi Deutsch-land GmbH . Pronase® was purchased from Roche Diagnostics GmbH . The pulmonary adenocarcinoma cell line A549 was kindly provided by Prof. Sergio Moya, CIC bioma GUNE, San Sebastian, Spain. The human normal bronchial epithelium cell line BEAS-2B was purchased from Sigma-Aldrich Chemie GmbH . Fetal bovine serum (FBS) was purchased from Biochrom GmbH , RPMI 1640 was purchased from Corning New York, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo EU GmbH . Lysotracker® Deep Red was purchased from Invitrogen . Doxorubicin hydrochloride (DOX) was purchased from Cayman chemical . Human serum albumin solution  was purchased from Grifols Deutschland GmbH . Dimethyl sulfoxide (DMSO), ethylene diamine tetraacetic acid disodium salt  at room temperature. Then, 0.05% of HSA solution was added to the suspension and incubated for further 5 min under stirring to prevent agglomeration of the particles. The suspension was centrifuged and the particles were washed twice (3000× g for 3 min). The resulting particle suspension was adjusted to a volume concentration of 30% and incubated with 10 mg/mL DOX in 50% DMSO for 1 h. After the incubation, the supernatant was collected and the particles were washed with 0.9% NaCl (3000× g for 3 min) until the supernatant became colorless. The resulting particles (30% volume concentration) were incubated with 0.1% GA solution for 1 h at ambient temperature allowing the cross-linking reaction to perform in the formulated submicron particles. The remaining unbound GA was quenched by incubation with 0.08 M glycine and 0.625 mg/mL NaBH4 for 30 min. The dissolution of the MnCO3 templates was performed by incubation with 0.5 M of EDTA at room temperature for 30 min. The obtained DOX-loaded HSA particles (DOX-HSA-MPs) were centrifuged, washed three times  and finally suspended in sterile 0.9% NaCl. The control HSA-MPs were fabricated following the same procedures without doxorubicin incubation.DOX-HSA-MPs, were fabricated by a slightly modified CCD technique as previously described ,23,24 F. Equal vt) and the doxorubicin amount determined in the supernatant after absorption and after each washing step (ΣDOXf). The entrapment efficiency (EE%) was calculated according to the following Equation (1):The amount of entrapped doxorubicin in DOX-HSA-MPs was calculated as the difference between the total amount of applied doxorubicin  at 480 nm. The calibration curve is given as The size and zeta potential of the DOX-HSA-MPs and HSA-MPs resuspended in PBS were measured by dynamic light scattering using a Zetasizer Nano ZS  at 25 °C. All measurements were performed in triplicate and the mean values were taken as the result.The DOX-HSA-MPs and HSA-MPs were observed by SEM . The samples were prepared by applying a drop of the particle suspension on a glass slide and then dried overnight at room temperature. Afterward, the samples were sputtered and coated with gold. SEM images were conducted at an operation voltage of 15 kV.®4 Bruker, Berlin, Germany). The samples were diluted in water and spread on a clean coverslip. The drop was completely dried after incubation at 30 °C for 1 h and thereafter the cover slide was stored in dry conditions (air) for 4 days at 24 °C. The images of the particles were then taken (dry state). For the wet stage images the clean cover slip was covered with poly-l-ornithine, rinsed with water and dried by nitrogen flow. The samples were dropped on the coverslip and incubated for 30 min. Afterwards the samples were thoroughly washed and imaged in water. The JPK Data Processing software  was used to analyze the obtained images.The morphology of HSA-MPs and DOX-HSA-MPs were investigated using AFM  equipped with a 100× oil immersion objective .® Deep Red were imaged using excitation wavelength 633 nm (HeNe laser) and emission long pass filter 650 nm. For intracellular co-localization of DOX-HSA-MPs and Lysotracker® Deep Red the particle fluorescence was detected using a band pass filter 530–600 nm.Images of autofluorescence of HSA-MPs and intrinsic DOX-fluorescence in DOX-HSA-MPs were taken using excitation wavelength 488 nm (Ar laser) and a long pass filter 505 nm. Cells additionally stained with Lysotracker® were placed into 2 mL microcentrifuge tube and incubated under shaking at 37 °C. At predetermined time intervals, the samples were centrifuged  and the supernatant was collected for DOX quantification using a microplate reader  at 480 nm. The release experiments were performed in triplicate, and the results were presented as mean ± SD. In parallel, the particles size was measured (Zetasizer Nano ZS).For in vitro drug release, DOX-HSA-MPs were resuspended in two different media at a volume concentration of 3%. (i) PBS pH 7.4 and (ii) PBS pH 7.4 containing 0.2 mg/mL PronaseTM) supplemented with 1% FBS. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 incubator .The cell line A549 is often used as an in vitro model of a lung cancer. Since albumin is endocytosed by alveolar epithelial cells via gp-60 albumin receptor ,29,30,312. Following these incubation times 10 µL of CCK-8 solution was added to each well and the plates were placed in the incubator for 1 h. Then the metabolic activity of the cells was assessed measuring the optical density at 450 nm using a microplate reader . Percentage of metabolic activity was calculated by the mean absorbance of the samples at each incubation time point. The mean absorbance of the control cells at 24 h was set to 100% and used as a reference value. Afterwards, statistical evaluation of the data was performed using an analysis of variance (two-way ANOVA). Tukey’s multiple comparisons were used to compare the significance of differences between each sample and the control. A value of p < 0.05 was considered statistically significant.In vitro colorimetric determination of cytotoxicity was assessed using Cell Counting Kit 8 (CCK-8). This assay kit measures the metabolic activity of dehydrogenases within the viable cells. The cells were seeded into 96-well culture plate at a concentration of 1000 cells/100 µL medium in each well and pre-incubated for 24 h to allow them to adhere. After this period, HSA-MPs and DOX-HSA-MPs were added to the cells at concentrations of 1000; 5000 and 10,000 particles/cell and incubated further for 24, 48 and 72 h at 37 °C, 5% CO5 cells/well in 24-well cell culture plates and incubated in a 5% CO2 humidified incubator at 37 °C for 24 h. The HSA-MPs and DOX-HSA-MPs particle suspensions (1000 and 5000 particles/cell) were then added and incubated for further 24 h. Following the incubation time, the cells were washed and fixed with 4% paraformaldehyde in PBS, washed again with PBS and incubated with a 0.25% trypsin solution for 5 min to harvest. After trypsinization, 1mL PBS was added per well and the total cell suspension was transferred for later reading. The percentage of cells with an uptake of particles was determined by flow cytometry  using the autofluorescence of the glutaraldehyde cross-linked protein for the HSA-MPs and the intrinsic fluorescence of DOX for the DOX-HSA-MPs, which was detected in the Allophycocyanin (APC)-channel (excitation 633 nm/emission > 660 nm). The uptake was additionally confirmed by Confocal Laser Scanning Microscopy (CLSM).To investigate the uptake of particles, A549 cells were plated at a density 1×10® Deep Red 30 min before the end of incubation time. Then the cells were washed with PBS, fixed with 4% paraformaldehyde and observed by CLSM.For intracellular localization of the HSA-MPs and DOX-HSA-MPs, the cells were additionally stained with 50 nM Lysotracker3 as a template is shown in 3-HSA-MPs mainly due to electrostatic and hydrophobic interactions. DOX can bind strongly to HSA via hydrophilic, hydrophobic contacts and hydrogen bonds [3 template is dissolved with EDTA and the final DOX-HSA-MPs are obtained.The fabrication process of DOX-HSA-MPs using MnCOen bonds ,32. AddiThe dynamic light scattering measurement of MPs in PBS demonstrated an average size of HSA-MPs and DOX-HSA-MPs in the range between 700 to 800 nm . No statistically significant differences of the hydrodynamic diameter of both particle types were found . In addiThe zeta potential values of HSA-MPs and DOX-HSA-MPs measured in PBS were not significantly different (−13.4 ± 0.8 mV for both particle types). This result implies that the positively charged DOX was not presented on the surface but rather immobilized inside the porous HSA-MPs.3 as a template.HSA-MPs A1 are viThe particles were further investigated by atomic force microscopy (AFM). v/v) DMSO in water and added to concentrated particle suspension (30% v/v). The entrapment efficiency for doxorubicin under these conditions was 25 ± 1.5% which corresponds to 7.25 fg DOX per particle.A high drug entrapment in drug carriers does not only reduce the necessary clinical dose, but also decreases the toxic side effects and improves the therapeutic effect ,34. In o® (0.2 mg/mL) was performed. As shown in ®. Simultaneously the particle size during the release experiment was also assessed , no significant differences between the metabolic activity of the cells cultured with both types of particles and that of the untreated cells were found . HoweverIn parallel, the DOX-HSA-MPs were also cultured with the non-cancerous cell line BEAS-2B derived from normal human bronchial epithelium. Here, also 1000, 5000 and 10,000 particles were added per seeded cell. The CCK-8 assay resulted in no significant differences between the cells cultured with DOX-HSA-MPs and untreated cells .Our particles consist of albumin, which is known to have an impact on the metabolic activity, cell proliferation and survival of cells by an interaction with such as co-factors, hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species .In the samples where the A549 cells were cultured for 72 h with HSA-MPs the metabolic activity was increased in comparison with that of the untreated cells. This could be understood as an extra nutrition that the cells receive after the uptake and digestion of the particles. In case of the DOX-HSA-MPs, the digestion leads to the release of DOX which interferes with the DNA  and decrN-acetyltransferase 1 (GNA1) causing proliferative delay and cell adhesion defects in A549 cells and leading to the superior drug effect of Abr [Our results are in concordance with a previous report about Abraxane (Abr), which is an albumin nanoparticle-bound Paclitaxel (PTX), a chemotherapy agent. It has been highlighted that Abr exhibited a greater effect for the treatment of non-small-cell lung cancer compared to free PTX. The authors found that albumin downregulates the protein glucosamine 6-phosphate t of Abr .For the quantitative determination of the cellular uptake of HSA-MPs and DOX-HSA-MPs we applied flow cytometry. As already observed by CLSM, both types of particles can be visualized by their intrinsic fluorescence if excited at 488 nm. For flow cytometry, we applied the APC channel in order to avoid influences by intrinsic fluorescence of the cells, which possibly can occur after their fixation with formaldehyde and is more intense if excited with wavelengths in the blue range. To clearly demonstrate the cellular uptake, we first determined the mean fluorescence intensity and distribution of the fluorescence intensity inside the particle populations . It can In To determine the location of particles in the cell compartments, A549 and BEAS-2B cells were incubated with DOX-HSA-MPs (5000 particles/cell) for 24 h and studies by CLSM. The images of the A549 cells incubated with DOX-HSA-MPs demonstrate a strong uptake  color confirms co-localization of both fluorophores and therefore the co-localization of particles and lysosomes. This localization is important for the degradation of the particles by lysosomal enzymes and for the release of the drug from the degraded particles.It can be seen that two DOX-HSA-MPs are internalized by one of the imaged cells. The overlapping of the red (DOX) and blue  is as follows. The DOX-HSA-MPs are firstly taken up at cell surface via endocytosis. Then, lysosomes and endosomes fuse to form autolysosomes and the scavenged particles are digested by lysosomal enzymes (more than 60 different types of enzymes) . After d3 template. Remarkably, the release of DOX from the DOX-HSA-MPs was suppressed under physiological conditions (PBS pH 7.4). However, in the presence of proteolytic enzymes DOX was released to roughly 40% from the particles. The DOX-HSA-MPs showed higher efficacy inhibiting the metabolic activity of A549 cells at lower dose than free DOX after treatment for 3 days, which correlated well with their localization in the lysosomal compartment. In addition, no uptake and toxic effects of DOX-HSA-MPs were found when the particles were cultured with the non-cancerous cell line BEAS-2B. This suggests that our carriers are a highly promising drug delivery system for an alternative chemotherapy treatment of cancer.We have successfully loaded DOX into protein particles via CCD technique. The DOX-HSA-MPs exhibited a submicron size with negative zeta potential. The DOX entrapped into protein particles around 25% which is likely due to hydrophilic, hydrophobic contacts and hydrogen bond between DOX and albumin in the MnCO


Despite the use of a high sensitivity assay, IL-8 reached detectable values only in 24% of IPF patients. EBC analysis after six months of treatment with pirfenidone did not reveal any differences in the cytokine levels. The change in EBC vascular endothelial growth factor A (VEGF-A) correlated with the change in the 6 min walk distance . We conclude that a six-month treatment with pirfenidone did not significantly change the EBC cytokine profile. Our findings support the potential usefulness of VEGF-A as a marker in IPF. The low EBC IL-8 level in patients with IPF is a novel finding which needs confirmation in larger studies.The mechanism of action of pirfenidone in idiopathic pulmonary fibrosis (IPF) has not been fully elucidated. To offer additional insight, we evaluated the change in the cytokine profile in exhaled breath condensate (EBC) following a six-month treatment with pirfenidone in patients with IPF. EBC concentrations of interleukin (IL)-6, IL-8, IL-15, TNF-α and VEGF-A were assessed with ELISA and compared at baseline and after six months of pirfenidone treatment. Twenty-nine patients with IPF and 13 controls were evaluated at baseline. With the exception of IL-8 concentration, which was lower in patients with IPF when compared to controls ( Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrosing interstitial lung disease of unknown etiology, which occurs primarily in older adults. Although the trigger for IPF is unknown, lung fibrosis in IPF seems to be regulated by a complex network of mediators ,2. It isThere are limited treatment options in IPF to date. Systemic steroids and immunosuppressive drugs had very modest efficacy, did not improve outcomes and increased the risk of death . In receExhaled breath condensate (EBC) is a noninvasively collected matrix from the respiratory tract, which contains various inflammatory mediators. Majewski et al. demonstrated that interleukin 25 and 33 are detectable in EBC from patients with IPF and that their concentrations show some significant correlations with lung function in these patients . We hypoThis prospective, observational study was a part of a larger project and included consecutive subjects with IPF who were qualified for the antifibrotic treatment. The study participants were recruited from the outpatient clinic of the Department of Internal Medicine, Pulmonary Diseases and Allergy, the Medical University of Warsaw between October 2017 and February 2019. The inclusion criteria for subjects with IPF were as follows: definite IPF diagnosis according to the 2011 ATS/ERS statement , forced All IPF subjects received antifibrotic therapy as prescribed by the attending physician. Demographic data, information on smoking history, symptoms and comorbidities were collected. The GAP index and staging system were used to estimate the mortality risk . Lung fuEBC samples were collected using the TURBO-DECCS 09 system  according to the manufacturer’s instructions and the ERS recommendations . The samThe selection of the investigated cytokines was based on our previous pilot study performed in patients with IPF in which the expression of the following cytokines was assessed on the multiplex bead-based cytokine assay: IL-1β, -1Ra, -5, -6, -8, -10, -15, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFNγ), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1 alpha (MIP-1α) and beta (MIP-1β), platelet-derived growth factor-beta (PDGF-β), TNFα, VEGF . Of thesp < 0.05.Continuous variables were described by median and interquartile range (IQR). Categorical variables were presented by number and percentage values. Statistical analysis was conducted using Statistica 13.3  software package. In the first step, basic descriptive statistics were calculated, and groups were compared with Fisher’s exact test. The differences between continuous variables were tested using the Mann–Whitney U-test for independent groups and the Wilcoxon signed-rank test for dependent groups. The strength and direction of the relationship between two variables were measured with Spearman‘s rank correlation coefficient. The level of significance in this study was assumed to be Twenty-nine patients with IPF (median age 73 (68–78) years, 83% male) and 13 control subjects (median age 62 (61–76) years, 69% male) were initially enrolled. There were no differences between the groups regarding baseline demographic characteristics. The proportion of current smokers was higher in controls, but the overall burden of smoking was similar in both groups. IPF patients had a significantly lower FVC than control subjects .Twenty-seven (93.1%) patients qualified for treatment with pirfenidone and two (6.9%) for nintedanib. This disparity resulted from the fact that, in Poland, pirfenidone has been reimbursed by the National Health Fund since January 2017, while nintedanib was approved for reimbursement approximately one year later, in March 2018, and has been available in our department since July 2018. Among the 27 patients on pirfenidone, 20 continued the treatment until the follow-up after six months, four died, and three ceased pirfenidone treatment because of adverse effects . The twoDetectable levels of IL-8 were found in 24% (7/29) and 77% (10/13) samples, of IL-6 in 79% (23/29) and 62% (8/13), and of VEGF-A in 76% (22/29) and 77% (10/13) of the samples from patients with IPF and controls respectively. A lower proportion of detectable values was found for TNF α: 10% (3/29) and 0% (0/13) for patients with IPF and controls, respectively, while the levels of IL-15 were under the limit of detection in all the analyzed EBC samples in both groups.p = 0.005). EBC IL-6 (0.09 (0.05–0.12) and 0.05 (0.0–0.13) pg/mL respectively, p = 0.586) and VEGF-A (4.69 (4.27–5.96) and 4.5 (4.46–5.62) pg/mL respectively, p = 0.837) did not differ between these groups; the levels of TNF-α and IL-15 were not compared due to the small number of samples with detectable concentrations of these cytokines (The EBC concentration of IL-8 was significantly lower in patients with IPF compared to controls (0.0 (0.0–0.0) and 0.65 (0.4–1.79) pg/mL respectively, ytokines .p = 0.020 and TLC % of predicted value , as well as positive correlation between EBC VEGF-A levels and the change of distance in 6MWD (m)  in patients with IPF. A positive correlation between IL-8 levels and BMI was found in both groups . We did not find any correlations between IL-6, IL-8 or VEGF-A levels and age, smoking exposure, time since smoking cessation, TLCO % of predicted value, delta SpO2 in 6MWT.We found a negative correlation between EBC VEGF-A levels and FVC % of predicted value .There was a negative correlation between EBC IL-6 and TNF-α, as well as a positive correlation between EBC IL-6 and VEGF-A  had EBC collection at baseline and after six months of treatment. Except for a significantly lower BMI after six months of treatment, the basic characteristics of the patients and their lung function did not change significantly in the follow-up period .p = 0.184); 0.0 (0.0–0.0) vs 0.0 (0.0–0.04) pg/mL for IL-8 (p = 0.441), and 4.9 (4.27–7.12) vs 6.54 (0.0–10.92) pg/mL for VEGF-A (p = 0.653) at baseline and after six months of treatment (There were no differences between cytokine concentrations at baseline and after six months of pirfenidone treatment in patients with IPF: 0.09 (0.06–0.13) vs 0.04 (0.0–0.14) pg/mL for IL-6 (reatment .p = 0.045) and with the change of IL-6 level , but not with the change in GAP score, BMI or pulmonary lung function. We found no significant correlations between changes in EBC IL-6 and IL-8 levels and changes in GAP score, BMI, FVC, TLC, TLCO and 6MWD. The change in EBC VEGF-A levels correlated with the change in 6MWD (m)  and a higher BMI after six months in those who had an increase in VEGF level (29.0 (26.1–30.3) vs. 26.0 (22.2–27.3) kg/m2, p = 0.048), no significant differences were found between the groups  patients, while in seven out of 20 (35%) patients, both markers decreased after treatment. These two groups did not differ in terms of clinical and pulmonary function data .Our study aimed to assess the changes in the EBC cytokine profile following a six-month treatment with pirfenidone in patients with IPF. We found a limited utility of ELISA measurements for IL-15 and TNFα in EBC from these patients. We showed that EBC concentrations of IL-8 are significantly lower in nontreated patients with IPF when compared to controls. Finally, we did not demonstrate significant changes in the EBC concentration of IL-6, IL-8 and VEGF-A during a six-month treatment with pirfenidone in these patients. This is one of the few studies analyzing the usefulness of EBC in monitoring treatment response to antifibrotic agents in patients with IPF and, to our knowledge, the first study to assess IL-6, IL-8, IL-15, TNF-α and VEGF-A in EBC from patients with IPF.Although a previous report showed that EBC analysis might modestly contribute to the diagnosis and monitoring of IPF , some auIn the present study, we assessed levels of these cytokines by ELISA technique. In a certain proportion of samples, they were below the assay detection limit. This particularly referred to IL-15, and TNFα as high sensitivity assays for these cytokines were unavailable, which precluded their further analysis. The comparison of the EBC cytokine concentrations between nontreated patients with IPF and controls showed a significantly higher EBC concentration of IL-8 in the latter. This is a rather unexpected finding, as it is not in line with earlier observations that almost uniformly support the involvement of IL-8 as a promoter of fibrogenesis ,29,30. ETo our knowledge, there have been no reports on IL-8 levels in EBC from patients with IPF to date. Not only did we find lower EBC IL-8 in patients with IPF when compared to controls, but also IL-8 reached detectable concentrations in 24% of these patients (vs. 77% controls) despite the use of a high sensitivity assay. This indicates that our finding may not be incidental and needs confirmation in larger studies. With the missing data on IL-8 in EBC, we currently find it difficult to explain. One of the potential reasons for the discrepancy between our results and the findings of other authors may be associated with the varying biomarker concentrations in different body compartments. This has been well documented in samples from patients with chronic obstructive pulmonary disease or asthma ,33. RepoA six-month treatment with pirfenidone did not significantly change the EBC concentrations of IL-6, IL-8 and VEGF. This is consistent with earlier findings on other EBC cytokines . There aWe did not observe any significant changes in lung function over the treatment period what may be interpreted as a sign of disease stability. In the study by Yoshikawa et al., an FVC decline of ≥10% and/or a TLCO decline of ≥15% of baseline was observed in 34.7% of the investigated group after six to nine months of observation . MajewskThere are several limitations of the study that need to be acknowledged. First, the diagnostic performance of cytokine measurements in EBC was relatively low and thus precluded all the planned analyses. However, our study was also exploratory, and one of our aims was to assess the feasibility of the selected cytokine measurements in EBC. Our findings confirm that conducting research with the use of EBC is challenging and highlight the need for high sensitivity measurement methods for EBC analysis. This may be the plausible explanation for the low diagnostic performance of the applied ELISA kits for IL-15 and TNF-α in our cohort as we did not use high sensitivity assays for these cytokines—to our knowledge such assays were not commercially available at the time of the study. Due to the particularly small sample volumes (as with EBC) no measurements were performed to normalize concentrations for dilution between subjects.On the other hand, this was also the case with VEGF-A and not only detectable concentrations of this marker were found in a considerable proportion of the investigated subjects but also some significant correlations between EBC VEGF-A and IPF clinical data were demonstrated. The second important limitation of this study is the disproportion between the number of patients treated with pirfenidone and those treated with nintedanib. This, however, was a result of the initial inaccessibility to treatment with nintedanib during the investigation period caused by the internal regulations of the Polish National Health Fund and the time interval between approval for the reimbursement of pirfenidone and nintedanib for patients with IPF. Therefore, to assure the maximal homogeneity of the investigated group and the credibility of our results, the analysis of post-treatment data was limited to patients treated with pirfenidone.Some differences in the characteristics of the investigated groups may also be regarded a drawback. Subjects from the control group tended to be younger; this was a result of significant difficulties in the recruitment of elderly subjects without lung disease who would voluntarily undergo respiratory investigations for research purposes. The difference in smoking status was negligible as the number of packyears did not differ between the groups. The differences in the incidence of comorbidities may result from the higher morbidity in IPF patients as compared to the general population ,45, therTo conclude, our findings provided some new data supporting the potential usefulness of VEGF-A as a marker in IPF. A six-month treatment with pirfenidone was not associated with a significant change in the tested cytokine profile of EBC. The low EBC IL-8 level in patients with IPF is a novel finding which needs confirmation in larger studies.


Supplemental Digital Content is available in the text methyltetrahydrofolate reductase (MTHFR) and chronic kidney disease (CKD) but there were no studies with a decisive conclusion. Furthermore, the high heterogeneity among different populations is not yet interpreted.Previous meta-analyses have explored the association between the C677T polymorphism of This study used trial sequential analysis (TSA) to evaluate whether the nowadays conclusion supported by current cumulative samples. We also applied case-weighted meta-regression to explore the potential gene–environment interactions.For the first stage of this study we conducted a case–control study involving 847 dialysis patients from 7 hemodialysis centers in Taipei during 2015 to 2018 and 755 normal controls from a health center in the Tri-Service General Hospital. The second stage combined the results from the first stage with previous studies. The previous studies were collected from PubMed, EMBASE, and Web of Science databases before January 2018.MTHFR C677T appeared to have a protective effect on end-stage renal disease compared with the C allele . However, the meta-analysis contradicted the results in Asian . The same analysis was also applied in Caucasian and presented similar results from Asian . The TSA showed our case–control study to be the decisive sample leading to a null association among Asian population. The high heterogeneity (I2 = 75%) could explain the contradictory results between the case–control study and the meta-analysis. However, further case-weighted meta-regression did not find any significant interaction between measured factors and MTHFR C677T on CKD.From the case–control study, the T allele of High heterogeneities were found in both Caucasian and Asian, which caused the null relationship in meta-analysis while there were significant effects in individual studies. Future studies should further explore the high heterogeneity that might be hidden in unmeasured gene–environment interactions, to explain the diverse findings among different populations. Genetic factors such as ethnicity and familial inheritance play important roles in CKD. The heritability of CKD is 20% to 80%, and individual differences originate from genetic mutations.–10 Genome-wide association studies have found that many polymorphisms cause CKD.,12 Therefore, exploring CKD-related gene polymorphisms is important for reducing the burden of the disease.Chronic kidney disease (CKD) is an important issue in public health. The prevalence of CKD is approximately 10% around the world.Methyltetrahydrofolate reductase (MTHFR) is a key enzyme of 1-carbon cycle. It can convert inactive 5,10-methylenete tetrahydrofolate (THF) to active 5-methyl THF, which assists in converting cysteine to methionine. Homocysteine (Hcy), produced during the conversion process, is pro-inflammatory and causes vascular irritation, which increases the risk of atherosclerotic damage. The most common polymorphism in MTHFR is C677T (rs1801133). The C677T locates in exon 4 of MTHFR and causes a change from alanine to valine. A previous study found that this polymorphism changed the thermolabile of MTHFR and the concentration of homocysteine increased significantly.MTHFR C677T and CVD, a meta-analysis of randomized control trials (RCTs) found that folate supplements did not significantly decrease the risk of CVD. Many previous studies have researched the relationship between C677T and CKD but the results are inconsistent due to differences in ethnicity, lifestyle, and comorbidities among the study groups.–22 Several meta-analyses have also explored the relationship between MTHFR C677T and diabetic nephropathy (DN) since 2007.–31 However, no satisfactory consensus has been reached. Therefore, we applied a trial sequential analysis (TSA) proposed by Thorlund et al to evaluate whether the most recent conclusions were supported by the current cumulative samples.Although previous studies have reported an association between  This could be applied in the relationship between MTHFR C677T and CKD to explore the heterogeneity.Although the meta-analysis had a higher sample size compared with the traditional case–control study, the population heterogeneity may cause meaningless result. This was mostly due to potential gene–environment interactions. Gene–environment interactions had not yet been explored in conventional meta-analyses owing to the difficulty in using summary data to analyze interactions. For this study, we developed a revised version of meta-regression, case-weighted meta-regression, which resolved the problem.The aim of this study was to evaluate whether the latest conclusions are supported by the current cumulative samples using trial sequential analysis (TSA). We also provided additional case–control samples to enhance the current evidence. Moreover, we applied case-weighted meta-regression to explore potential gene–environment interactions.22.12.1.1This study was approved by the institutional review board (TSGH-1-104-05-006) of the Tri-Service General Hospital (TSGH). Volunteers signed the consent form after the investigators had provided an explanation of the study.2.1.2Subjects in the case group were hemodialysis patients from 7 Taipei Dialysis Centers during 2015 to 2017. The exclusion criteria were as following:(1)dialysis period of < 3 months,(2)presence of cancers, and(3)insufficient blood samples.A total of 882 cases were included in the analysis. was used to calculate the estimated glomerular filtration rate (eGFR). The exclusion criteria were shown as following:Subjects in the control group were selected from volunteers who participated in a physical exam at the Health Management Centre at TSGH. The serum creatinine levels of volunteers were tested, and the Modification of Diet in Renal Disease (MDRD) formula(1)2,eGFR < 60 ml/min/1.73 m(2)presence of kidney-related diseases (such as positive proteinuria),(3)presence of cancers, and(4)insufficient blood samples.Finally, 755 subjects were included in the control group for the analysis.Demographic data including age, sex, diabetes, hypertension, body mass index (BMI) and blood biochemical parameters  were collected via questionnaire and medical records.2.1.3MTHFR C677T was genotyped by iPLEX Gold SNP genotyping. Inter- and intra-replication validation was used to assess the genotyping experiment quality. Inter-replication validation was repeated for 78 samples (∼5%), and the concordance rate was 100%.Medical technologists or nurses collected 5 mL of intravenous blood samples from each of the volunteers. Genomic DNA from peripheral blood samples was isolated using standard procedures for proteinase K  digestion and the phenol/chloroform method.2.1.4t test. The control group was tested for representativeness using the Hardy–Weinberg equilibrium (HWE) test. Differences in genotype and allele frequencies between hemodialysis patients and healthy controls were tested using a χ2 test or Fisher exact test. ORs and 95% confidence intervals (CIs) for the risk of end-stage renal disease (ESRD) were calculated using logistic regression. Calculation of genetic polymorphism and ESRD risk was expressed using allele type, genotype, and dominant/recessive models. A p-value of < 0.05 was considered significant, and the Bonferroni correction was used for multiple comparison correction. R 3.4.2  was used for statistical analyses.Continuous variables of the general demographic data were expressed as mean and standard deviation using Student 2.22.2.1 Related terms of “MTHFR C677T” and “chronic kidney disease” were used to search the PubMed, EMBASE, and Web of Science databases for articles published up to December 31, 2018 Supplemental Digital Content (S2 Table). The language of the articles was limited to English. In addition, the publications included in the meta-analysis studies were manually examined to avoid the omission of important articles. The inclusion criteria were as following:The PRISMA checklist and Meta-analysis on Genetic Association Studies Checklist are described in Supplemental Digital Content (S1 Table).(1)the study design was case–control or cross-sectional;(2)2 ;CKD was defined according to the National Kidney Foundation as kidney damage by clinical diagnosis or a GFR < 60 mL/min/1.73 m(3)subjects in the control group had normal renal function;(4)MTHFR C667T; andthe article contained the detailed genetic distribution of the (5)the samples were aged >18 years.When the proportion of diabetic subjects in the case group in an article was 100%, the control group was selected from diabetic patients.2.2.2Two reviewers (G-rC and CL) independently extracted the literature data. The collected data included the last name of the first author, year of publication, country, race of the study population, male proportion, mean BMI, diabetes prevalence, hypertension prevalence, smoking prevalence, and gene distribution in the case and control groups.2.2.3MTHFR C677T polymorphisms and CKD. I2 calculated with the Cochrane Q test was used to assess heterogeneity. I2 > 50% indicated moderate-to-high heterogeneity. Egger regression and funnel plot were used to examine the symmetry after combination. Genetic models including allele type, and dominant and recessive models were used to calculate MTHFR C677T polymorphisms and risk of CKD. The random effects model was used to combine results.The data of all included articles were described using proportions or mean values where appropriate. The meta-analysis used ORs with 95% CI to examine the correlation between Two methods were used to explore the moderator effects in previous meta-analyses:(1)dividing different studies into high- and low-risk groups according to the distribution of moderators for subgroup analysis; and(2)applying meta-regression to analyze the correlation between effect size and the distribution of moderators.,41 Therefore, meta-regression is usually used for moderator effect analysis. However, the matched studies (such as gender- and age-matched studies) were the necessary condition to use traditional meta-regression for analyzing moderator effects. Lin et al have developed a revised version of meta-regression, known as case-weighted meta-regression, to break this limitation. Under the 2 assumptions, rare disease and independence between moderator and independent variable, the odds ratio (OR) of exposure to the disease tended to be close to the proportion of moderators in the case group. Therefore, it was better to extract data from the case group during the meta-analysis.In the first method, it is difficult to determine the cutoff point for the moderator, and this tends to increase the probability of false positives. and “meta” packages of R software version 3.4.2 were used. TSA was used to validate whether the meta-analysis results obtained a decisive conclusion. OR was used as an effect measure, and the random effects model was used to combine results. Zero event handling was set at 1. The degree of freedom was set at 2. TSA was used for stratification analysis based on race (Caucasian and Asian). Type 1 error was set at 0.05. Power was set at 0.8. Heterogeneity was set at 80%. A review of past literature showed that the OR of correlation between MTHFR C677T and CKD was around 1.2. The 1000 Genome database was used as a reference for minor allele frequency, which is 0.30 for Asians and 0.36 for Caucasians.Considering the high heterogeneity of the combined results, case-weighted meta-regression was applied to examine the source of heterogeneity in this study. Possible environmental factors  were included, and summary data from the case group were extracted to explore gene–environment interactions. The significance level of this study was set at 0.05. The “metafor"33.1P < .001), and the mean age in the control group was higher than that in the case group (P = .006). The case group showed a higher proportion of diabetic patients, a higher proportion of hypertensive patients, higher creatinine levels, higher fasting blood glucose levels, and higher triglyceride levels than the control group. In addition, the case group showed lower cholesterol level. Table MTHFR C677T genotype between the case and control groups. The gene frequency of the control group followed the Hardy–Weinberg equilibrium (P = .493). The distribution of the T allele in the control and case groups was 30.5% and 25.9%, respectively, and this difference was significant (P = .004).Table MTHFR C677T was a significant protective factor for ESRD [OR: 0.760 (95% CI: 0.639–0.904)] . The allele model was used to combine 34 populations, and the results showed significant differences [OR: 1.15 (95% CI: 1.02–1.30)] and high heterogeneity (I2 = 82%). However, the stratified analyses showed null associations in Asian [OR: 1.12 (95% CI: 0.96–1.30)] and Caucasian [OR: 1.18 (95% CI: 0.98–1.42)], respectively. No significant asymmetry was discovered between the articles. The forest and funnel plots are shown in Fig. The study identification process is shown in Fig. 3.3MTHFR C677T and all-cause CKD are not significantly related , and a decisive conclusion could be validated. Before adding our case–control samples, the cumulative sample size for the Asian population was 8021 , showing that 021 Fig. . After a3.42 = 75%) in Asian could explain the contradictory results between the case–control study and the meta-analysis. The overall analysis had shown a significant association, but stratified analyses showed a non-significant association in both the Asian and the Caucasian sample groups. Therefore, environmental moderators should be explored to explain the heterogeneity. Table MTHFR C677T and CKD . Case-weighted meta-regression was used to analyze the race, sex, BMI, hypertension, DM, and smoke. However, there was no significant gene-environmental interaction between all measured factors and MTHFR C677T.The high heterogeneity  in the Asian sample could explain the contradictory results between the case–control study and the meta-analysis.The case–control study found T allele is a significant protective factor for ESRD after adjusting for age, sex, comorbidities, and other confounding factors. Although these results are consistent with those reported by of Bloudickova et al, Even we had conducted the genome wide association study on a lot of complex phenotype such as height, diabetes, and cholesterol, we still only can find a small part of significant polymorphism and just can explain a little part of each phenotype. This phenomenon is still not solved now in complex phenotype. Chronic kidney disease is clearly a complex disease, which might be affected by multiple factors including ethnicity and family history of disease. In the best of our knowledge, the genome wide association study with large sample also only explained 2% variance in chronic kidney disease. Therefore, the non-significant finding was common. The significant findings in individual studies but non-significant results in larger study might be due to the lack of control for population stratification bias. The high study heterogeneity of our meta-analysis might be due to this issue, and we considered the studies using mixture population was not suitable. We considered the future study should be conducted by whole genome survey process and use the related method for correcting this issue.The inconsistent result between proposed homocysteine related mechanism and our non-significant result might be explained by missing heritability issue. The term “missing heritability” was first proposed by Manolio in 2009.MTHFR C677T, suggesting that there may be further unmeasured factors to be discovered. Additional studies exploring the effects of gene–environment interactions on CKD are warranted. Ma et al proposed a significant interaction between MTHFR C677T and smoking in people with type 2 diabetes or DN. Among those who smoked, the risk of DN was higher in subjects with the T allele . Another large-scale cross-sectional study in Japan found no significant interaction between MTHFR C677T and blood folate concentration on CKD. Further stratified analysis found that the risk of developing CKD was higher in subjects with the TT genotype and low blood folate concentration . Regarding drug–gene interactions, many RCTs have showed significant interactions between MTHFR C677T and folate supplements. In subjects with the TT genotype, the Hcy reduction effects of folate supplements were significant, although a meta-analysis of large-scale RCTs denied the presence of such interactions.This meta-analysis did not detect significant interactions between the measured environmental factors and  found that polymorphism at MTHFR C677T and another locus (A1298C) increased risks of micro-albuminuria and macro-albuminuria . Jafari et al found significant interactions between endothelial nitric oxide synthase (eNOS) G894T and MTHFR C677T in type 2 diabetes, and where both T allele existed, the risk of developing macro-albuminuria was higher . eNOS encodes nitric oxide synthase, which plays an important role in regulating vascular endothelial function. MTHFR C677T polymorphism increases Hcy concentration and disrupts vascular endothelial cell function. Therefore, we suggest further studies investigate the gene–gene interaction between these 2 genetic factors and MTHFR C677T. However, the 2 aforementioned articles had low sample sizes, and a larger one may be required to validate these interactions.In addition to gene–environment interactions, other studies have explored the effects on the risk of CKD of gene–gene interactions. Rahimi et alMTHFR C677T and CKD in PubMed, EMBASE, and Web of Science (including this study) databases and included 19,734 subjects. In addition, TSA analysis found that a decisive conclusion could be established and the samples in this study were critical evidence. Secondly, due to limitations in summary data for conventional meta-regression analysis, sex, age, and other variables could not be used for interaction analysis. In this study, case-weighted meta-regression was firstly applied for interaction analysis, which not only solved the problem of insufficient power in individual articles but also detected potential interactions in meta-analysis. Thirdly, the Newcastle–Ottawa Scale was used to perform a complete article quality evaluation, and whether article quality affected the combined results was investigated. Although there was no effect of this factor on the combined results, this approach enhances the reliability of this study. Finally, our meta-analysis could prevent errors compared with previous studies. Our study excluded articles with incomplete genotype information and used the allele model to combine results. Egger regression was not significant for Asians and Caucasians (P > .05). In previous meta-analyses, the fixed effects model was used for combination if heterogeneity was not significant (P ≥ .1) and the random effects model was used if heterogeneity was significant (P < .1).–125 This study only used the random effect model to combine results, and could avoid the serious bias that can be caused when heterogeneity is used to select a model.Our study has the following strengths. To begin with, we included 33 English articles regarding the correlation between MTHFR C677T and CKD could not be explored. Third, many of the articles did not provide the complete distribution status of the moderators , which decreased the powers. We suggest future case–control studies should provide detailed information on these factors.This study has 3 limitations. First, because individual data could not be obtained in the meta-analysis, the possibility of ecological fallacy cannot be excluded. Although this is a limitation of the meta-analysis, many variables were included in this study and the correlation between these variables was explored. Therefore, the possibility that the study conclusion will be affected by ecological fallacy is extremely low. Second, data on the diet and folate intake status are limited. Therefore, the potential effect of folic acid intake on the association between 5MTHFR A1298C. More specific stratified analysis based on heterogeneity may help us to understand the real relationship between MTHFR C677T and CKD.In conclusion, high heterogeneities were found in both Caucasian and Asian, which produced a null relationship in meta-analysis but significant effects in individual studies. However, after using the case-weighted meta-regression, all the measured factors still cannot explain the source of heterogeneity. Future studies should further explore the high heterogeneity, which might be hidden in unmeasured gene–environment interactions, to explain the diversity findings among different populations. The gene–gene interactions should also be considered such as eNOS G894T and H-L C, M-C T, C-C K, W-T C, S-L S conceived and designed the experiments. H-L C, W-T C, H S performed the experiments. H-L C and D-JT analyzed the data. H-L C, P-J H, C-C C, S-L S provided the reagents, materials, and analysis tools. H-L C and G-R C wrote the paper. H-L C, D-J T, Y-H C, W-T C, S-L S were responsible for the critical review and providing comments. H-L C, S-LS and D-JT were responsible for modifying the manuscript.


The aim was to investigate the effectiveness of pegylated liposomal doxorubicin (PLD), beta-carotene, and a combination of PLD and beta-carotene on JAR and JEG-3 human choriocarcinoma (CC) cell lines for the treatment of CC.JAR and JEG-3 cells were cultured. PLD and beta-carotene trial groups were determined with different doses . Drugs were administered to cultures simultaneously, and 72 hours later the cells were detached using trypsin-ethylenediamine tetraacetic acid solution. The percentage of apoptotic cells was determined by flow cytometry after annexin V staining. One set of the supernatant was collected before trypsin application to investigate beta-human chorionic gonadotropin (β-hCG) and hyperglycosylated hCG (H-hCG) levels. Statistical analyses of the apoptotic ratios were performed using Shapiro-Wilk, Kruskal-Wallis and Mann-Whitney U tests.Apoptosis increased in JAR and JEG-3 cultures after treatment with all doses of PLD (p<0.05). A single application of each beta-carotene dose increased apoptosis in JAR cells (p<0.05) but had no apoptotic effects on JEG-3 cells. In the PLD and beta-carotene combination group, apoptosis increased in both JAR and JEG-3 cells (p<0.05).To our knowledge, this is the first investigation of the effectiveness of PLD, beta-carotene, and PLD + beta-carotene combination therapy in two different CC cell lines. PLD is a promising chemotherapeutic drug, and beta-carotene can be used as a novel non-chemotherapeutic agent for treatment of CC. Based on the results of this study, vitamin A supplementation may have promise as a preventive measure. However, these data need support from animal experiments and clinical trials. Gestational trophoblastic disease (GTD) appears in the fetal chorion and involves a variety of interrelated diseases. The effects of GTD range from benign hydatidiform moles (HM), which usually resolve spontaneously, to life-threatening. GTD usually develops from HM, but it has been observed in aborted, term, and ectopic pregnancies. Choriocarcinoma (CC) is the most aggressive histologic type of GTD . The mosThere are many medical treatments for CC depending on the stage of the disease. Multi-agent chemotherapy protocol is the preferred treatment method if hCG levels increase during treatment, if metastasis develops, or a resistance develops to sequential single-agent chemotherapy protocol . The curDoxorubicin is an anthracycline antibiotic which intercalates between base pairs in the DNA helix, thereby preventing DNA replication and ultimately inhibiting protein synthesis . PegylatBeta-carotene is a naturally-occurring retinol (vitamin A) precursor obtained from certain fruits and vegetables with potential antineoplastic and chemopreventive activities. It is particularly protective in chemical carcinogenesis by taking part in the detoxification of peroxide radicals . It was The options that can be used against multiple drug resistance in the treatment of CC are limited in the literature. Therefore, the aim of this study was to investigate the effects of PLD and beta-carotene and treatment efficacy on cell culture CC models using JAR and JEG-3 cell cultures in order to provide more effective treatment methods by providing a new perspective in CC treatment.2 incubator in 75 cm2 flasks.This study was planned as pre- and post-test study. JAR and JEG-3 cell culture lines were obtained from the American Tissue Type Culture Collection. All cell cultures were maintained and cultured in Roswell Park Memorial Institute (RPMI) - 1640 medium  supplemented with 10% heat-inactivated fetal calf serum,  penicillin streptomycin, and L-glutamine  in a 98% humidified, 5% CO2 atmosphere at 37 °C in a Nuve EC 160 COPLD was purchased from Sigma-Aldrich Chemie GmbH, Germany, prepared in dosages of 1, 2 and 5 μg/mL by diluting in dimethyl sulfoxide (DMSO) , and diluted in RPMI-1640 to a maximum concentration such that DMSO formed less than 1% of the mixture.Beta-carotene was purchased from Sigma-Aldrich Chemie GmbH, Germany, prepared in dosages of 1, 5 and 10 μg/mL by diluting in DMSO, and diluted by RPMI-1640 to a maximum concentration such that DMSO formed less than 1% of the mixture.The dosages used in the PLD and beta-carotene trial groups were as follows:Single drug trial: PLD 1, 2 and 5 μg/mL; beta-carotene 1, 5 and 10 μg/mL.Combined PLD and beta-carotene drug trial: PLD 1, 2 and 5 μg/mL; and beta-carotene 5 μg/mL.Drugs were administered to the cells simultaneously, and 72 hours after drug administration, the cells were detached from the bottom of plate by using trypsin ethylenediamine tetraacetic acid (EDTA) solution. The degree of apoptosis was determined by flow cytometry (FCM). The supernatant was collected before trypsin application from one set of samples from each experiment and stored at -80 °C frozen to investigate H-hCG levels. H-hCG levels were investigated using an immunoenzymatic method . All tests were repeated six times.The annexin V-binding assay is one of the most sensitive and widely used techniques to detect and distinguish between early apoptosis and late apoptosis, as well as between apoptosis and necrosis. Annexin V is a protein that binds preferentially to phosphatidylserine, which is located at the outer surface of the cell membrane. This feature allows apoptotic cells to be observed after marking them with a fluorescent agent such as fluorescein isothiocyanate (FITC) . The binEthics committee approval was received for this study from the Ethics Committee of Bülent Ecevit University Faculty of Medicine . No informed consent was obtained due to cell culture study.Statistical analyses of degree of apoptosis were performed with the SPSS, version 19.0 package program. Descriptive statistics related to continuous variables are given as median and range . The conformity of variances to normal distribution was evaluated through the Shapiro-Wilk test. The Kruskal-Wallis test was used in quaternary comparison of doses and the Mann-Whitney U test and the Mann-Whitney U test with Bonferonni correction were used in binary subgroup comparison between doses. In all statistical analysis in the study, comparisons below a p-value of 0.05 were accepted as statistically significant.The median apoptotic ratio in the control group, in which only DMSO was used, was 0.3% in the JAR cell culture and was 3.7% in the JEG-3 cell culture. The median apoptotic ratios after the application of 1, 2 and 5 μg/mL PLD were; 1.3, 1.4 and 2% in the JAR cells, respectively. The increase in the apoptotic ratio was statistically significant (p<0.05) , Graph 1After 72 hours, median β-hCG levels in JAR and JEG-3 cell cultures were respectively, 126 and 118 mlU/mL. Median H-hCG levels in JAR and JEG-3 cell cultures were respectively, 63 and 66 mlU/mL . After tThe median apoptotic ratios after the application of 1, 5 and 10 μg/mL beta-carotene were 0.35, 0.6, and 0.8% in the JAR cells, respectively. The increase in the apoptotic ratio was statistically significant (p<0.05) , Graph 2After the application of 1, 5 and 10 μg/mL beta-carotene to JAR cell lines, median β-hCG levels were: 107, 115 and 111 mlU/mL, respectively; median H-hCG levels were: 64, 68, and 56 mlU/mL, respectively . In JEG-The median ratios of apoptosis were 1.3, 1.4 and 2% after application of 1, 2 and 5 μg/mL PLD in JAR cell cultures, respectively; as mentioned above. With the addition of 5 μg/mL beta-carotene to those PLD doses, and after application of 5 μg/mL beta-carotene combined with 1, 2 and 5 μg/mL PLD, the median apoptotic ratios were 0.4%, 1% and 1%, respectively. This incremental increase of the combined doses were statistically significant in comparision with the control group (p<0.05), but the incremental increase in doses of PLD alone were statistically significant compared to 5 μg/mL beta-carotene combined with PLD (p<0.05) , Graph 3The median ratios of apoptosis were 16.1, 31.4 and 27.5% after application of 1, 2, and 5 μg/mL PLD in JEG-3 cell cultures, respectively; as mentioned above. With the addition of 5 μg/mL beta-carotene to those PLD doses, and after application of 5 μg/mL beta-carotene combined with 1, 2, and 5 μg/mL PLD, the median apoptotic ratios were 6.1%, 5.7% and 15.5%, respectively. This incremental increase of the combined doses were statistically significant in comparision with the control group (p<0.05), but the incremental increase when PLD was used alone were statistically significant in comparision to the combination of PLD at different concentrations and 5 μg/mL beta-carotene (p<0.05) , Graph 3Cell culture studies are frequently employed, especially in the evaluation of newer drugs. This is because it enables determining the effects caused by candidate drugs on molecular targets, which are common or are exptected to be effected by those drugs, and predicting the effects that the drug will have on target tissue. Recurrent tumor growth due to unpredicted changes caused by newly developed cancer drug components on cell phenotype may lead to unexpected results in clinical practice. The use of human monolayer cancer cell lines is fairly common and an important method for understanding the causes of these unexpected results obtained in clinical practice . JAR andVarious drugs have cytotoxic effects on cells through either necrosis or apoptosis or a combination of these. It is a preferred characteristic of drugs used or being developed to be used in cancer treatment that the cytotoxic effects function in apoptotic pathways, as one of the mechanism leading to cancer is disruption of the normal apoptotic mechanism of a pre-cancerous cell, and domination of anti-apoptotic signal pathways of the cell on apoptotic pathways. Thus, an aim of this study was to analyze the apoptotic effect due to the use of PLD and beta-carotene, alone or in combination. In this study, the degree of apoptosis of JAR and JEG-3 cells was identified with FCM.GTD is an interrelated group of tumors, characterized by abnormal proliferation of placental trophoblasts . Benign Complete molar pregnancy risk increases in those with dietary deficiency of vitamin A (carotene) and animal fat, while there is no increase in partial molar pregnancy incidence (9). CC is seen once in every 50.000 pregnancies . AdvanceAll forms of GTD are characterized by β-hCG increase due to the interrelated, heterogeneous structure of GTD, especially arising from trophoblastic epithelium of the placenta, and gestational tissue is responsible for pathogenesis of GTD. HCG is used as a biomarker for diagnosis and follow-up of the disease and response to treatment. H-hCG is a glycosylated variant of β-hCG with a larger side chain produced by invasive cytotrophoblast cells in pregnancy implantation, GTD and CC. An increase in H-hCG levels has been reported in testicular and ovarian germ cell tumors . Thus, tCole et al.  showed iPLD is a long-release formulation consisting of doxorubicin hydrochloride contained in pegylated liposomes. In breast cancer patients with increased cardiac risk PLD is administered and is also used in advanced ovarian cancer cases when platinum-based chemotherapy is unsuccessful and acquired immuno-deficiency syndrome with Kaposi sarcoma. Embedding doxorubicin into liposomes causes changes in pharmacokinetics and biodistribution and is reported to decrease toxic effects . SoinineEetezadi et al.  evaluateHanf et al.  evaluatePopadiuk and Power  showed tSaul et al.  showed tPariente et al.  evaluateSimilarly, it has been shown that in vitro melatonin potentiates cytotoxic and apoptotic effects of CIS and particularly 5-FU by stimulating MT3 receptor in the human colorectal adenocarcinoma cells HT-29) and HeLa cells . Cell vi and HeLaThere are studies showing that carotenoids are protective against head, neck, mouth, skin, lung and other malignancies and hematopoietic diseases. Furthermore, increased dietary intake of carotenoids  was associated with lower oesophagus cancer risk. Most of the in vitro studies recently focused on anti-carcinogenic mechanism of beta-carotene in lung, liver and blood cells. In some animal studies, alfa-carotene demonstrated a higher suppressor activity on liver, lung, skin and colon carcinogenesis compared to beta-carotene .Studies have shown that the molecular protective mechanisms of carotenoids in isolated human cell culture include:  stoppingDutta et al.  showed tWang et al.  indicateGloria et al.  in a celTo the best of our knowledge this is the first report presenting the effect of beta-carotene in combination with PLD on JAR and JEG-3 CC cells in vitro. When beta-carotene was administered alone and at increasing doses, its apoptotic effects on JAR cell significantly increased. However, its apoptotic effects on JEG-3 cell was not significant. When PLD was administered alone at increasing doses its apoptotic effects on both JAR and JEG-3 cells significantly increased. When increasing concentrations of PLD were combined with a fixed concentration of beta-carotene there was a significant increase in apoptotic effect in both JAR and JEG-3 cells.This is the first report, as far as we are aware, presenting the effects of beta-carotene and PLD in combination on JAR and JEG-3 human CC cell line models. The effects were assessed by measuring the concentrations of β-hCG and H-hCG. Apoptotic data showed that beta-carotene and PLD combination has a synergistic effect and may be a viable option for treatment of multiple drug resistant human CC. The results of this study suggest that vitamin A supplementation may have a preventive role in human CC in the future. However, these were in vitro studies and the results should be confirmed in animal trials and, if justified, clinical studies, as the effects of the drugs and the effects in combination may differ in in vivo systems.


Previous studies have shown that tuberculosis (TB) is a risk factor for chronic airflow limitation. Chronic obstructive pulmonary disease (COPD) is recognized as the result of chronic inflammation, usually related to noxious particles. Post-TB airflow obstruction and tobacco-related COPD have the same functional pathway characterized by persistent airflow limitation. We sought to compare the profile of 29 cytokines in the sputum of subjects with post-TB airflow obstruction and those with COPD related to tobacco.The forced expiratory volume in the first second (FEV1) and forced expiratory volume/forced vital capacity (FEV/FVC) ratio were lower in the COPD patients with the history of smoking compared to the post-TB airflow obstruction subgroup. The stages of the disease were more advanced in COPD / tobacco patients.p values of 0.003, 0.0001, 0.03, 0.0001 and 0.02 respectively. When the two COPD subgroups were compared, IL-1α, IL-6, TNF-α and IL-8 levels were higher in the COPD patients with the history of tobacco compared to the COPD patients with the history of TB with p-values of 0.031, 0.05, 0.021 and 0.016, respectively.Among the cytokines, IL-1α, IL-1β, MIP-1β, sCD40L and VEGF levels were higher in COPD patients, compared to the controls with COPD related to tobacco is more severe than post-TB airflow obstruction. The pathogenesis of post-TB airflow obstruction appears to involve the cytokines IL-1RA, IL-1α, IL-1β, IL-17, GRO and sCD40L, while COPD related to tobacco involves more cytokines. Chronic obstructive pulmonary disease (COPD) is a respiratory disease characterized by progressive airflow obstruction that is not fully reversible, with combined emphysema and chronic obstructive bronchitis . It is aCigarette smoking is known as the main risk factor of COPD . The defThe damage induced by cigarette smoke leads to the limitation of respiratory flow that induces emphysema and chronic bronchitis that are peculiar to COPD.Although smoking is the main cause of COPD , the samPost-TB airflow limitation is also common after TB treatment . InflammKnowledge of the inflammatory characteristics of COPD and/or post-TB AFO could help to better adapt the anti-inflammatory treatment of patients, and thus reduce the frequent phenomenon of corticoresistance . The purp values of 0.0023 and 0.0002, respectively. A multivariate analyzes indicated that spirometric data were not influenced by the sex and age, with the following results: for the sex, odd ratio (IC 95%) = 2.0 (1.05–10.71) and p = 0.54; for the age, odd ratio = 2.74 (1.15–109.3) and p value of 0.51.A total of 150 participants were recruited: 90 COPD patients and 60 clinically healthy non-smokers for the control group. The COPD patients consisted of 50 patients with a history of smoking (COPD/tobacco) and 40 patients who previously had TB (COPD/post-TB) , pro-inflammatory cytokines , chemokines  and growth factors . Other cytokines such as IFN-α, IFN-γ, IL-10, IL-12, MDC, PDGF, IL-15, IL-2, IL-4, IL-7 and RANTES were not detected in the sputum of either the COPD patients or the control group.p-values all lower at 0.05. IL-6 and TNF-α were higher in COPD patients with a history of tobacco compared to the post-TB airflow obstruction patients and to the control. GRO concentration was higher in COPD patients with anterior TB than in the control. There were no statistically significant differences in cytokine concentrations, such as IL-17, GM-CSF, G-CSF, MIP-1α and IP-10.Table p-values of 0.031; 0.049; 0.021 and 0.016 respectively, with no significant differences on the other cytokines , or this result could be linked to the fact that most COPD/post-TB subjects were at a less advanced clinical stage . Suleyman et al.  found thLevels of TNF-α were statistically significantly higher in the COPD patients with a history of smoking. Generally, the secretion of TNF-α in COPD is induced by tobacco smoke and maintained by the chronic inflammation process . TNF-α hMycobacterium tuberculosis is no longer present in the post-TB airflow obstruction patients, there is no real inducing element of TNF-α, which could justify its low concentration in COPD/post-TB patients.Since IL-17 concentrations in both subgroups of COPD patients were higher compared to the control group. During post smoking COPD, IL-17 plays a role in T cell proliferation, activation of fibroblasts, endothelials and epithelial cells, induces release of cytokines  and activation of neutrophils , 24. IL-Regarding MIP-1β, a higher concentration was noted in the COPD patients with a history of tobacco compared to the control group. MIP-1β is a chemokine that induces lymphocyte migration and recognizes CCR5 receptors in T cells and macrophages . Some stThe high expression of IL-8 in smoker patients has certainly been induced by cigarette smoke, and is maintained by chronic intoxication of the airways as is the case in the studies conducted by Becker and colleagues . StudiesSCD40L bind to CD40 of monocytes to promote their adhesion to the vascular endothelium, and to endothelial cells to activate them. Some studies have found high concentrations of sCD40L in certain diseases such as severe sepsis  and in cVEGF was strongly expressed in the sputum of COPD patients with a smoking history compared to the control group, and not expressed in the COPD patients with anterior TB. VEGF is a growth factor involved in angiogenesis (forming new preexisting vessel growths) in asthma. As in asthma, VEGF would be involved in the mechanism of bronchial vascular remodeling during COPD. Kanazawa et al.  found anThe objective of the study was to compare the profile of certain cytokines in COPD/tobacco and COPD/post-TB patients. We reached the following conclusions: COPD related to tobacco use is more severe than the post-TB form. The pathogenesis of COPD in patients with a history of smoking involves several cytokines in the local pathway such as IL-1α, IL-1β, IL-6, IL-17, TNF-α, IL-8, MIP-1β and VEGF. In contrast the pathogenesis of the post-TB form seems to imply fewer inflammatory markers with IL-1RA, IL-1β, GRO and sCD40L as local marker.Study participants were recruited from the Yaoundé Jamot Hospital (YJH) from February 2016 to July 2017. YJH is the main and biggest center in Cameroon specializing in the management of respiratory disorders. Participants constituted COPD patients and healthy non-smokers who served as controls. COPD participants were chosen among patients consulting at the YJH and who had been diagnosed by a pneumologist. COPD patients comprised two sub-groups: patients with a history of smoking (COPD/tobacco) and patients with post-TB airflow obstruction (post-TB AFO or COPD/post TB). The COPD/tobacco group had clinical signs of COPD during active smoking and they stopped to smoke once they understood that cigarette smoke was the cause of their disease. Post-TB AFO patients had previously one or more episode TB without smoking. Participants were eligible for the study if they gave written informed consent for participation, including consent for HIV testing. Patients were excluded if they had active TB, or were physically or mentally unable to perform a respiratory function test. The work received ethical clearance from the Cameroon National Ethical Committee of Research for Human Health (N° 2016/06/772/CE/CNERSH/SP).Once the pneumologist had diagnosed an eligible patient with COPD, the spirometric results of the patient were extracted from his/her medical record. The history of TB or smoking was also extracted from patients’medical records and confirmed by each patient. Spirometric measurements for the control group were done with the turbine pneumotachograph  following the American Thoracic Society standard to ensure that participants did not have any respiratory problems. Sputum samples were collected from each participant. Samples were transported at ambient conditions in a coolbox to the Center for the Study and Control of Communicable Diseases of the Faculty of Medicine and Biomedical Sciences, University of Yaoundé I. Upon receipt in the laboratory, cell counts  were performed after staining of the smear with May-Grunwald Giemsa and read by light microscopy. After the cell counts, the sputum was liquefied by mixing with phosphate buffered saline (PBS). The sputum was centrifuged at 2000 rpm for 10 min, and the supernatant was aliquoted and stored at − 80 °C until cytokine analyses. The cytokine analyses were performed by the Luminex technique at Stellenbosch University Immunology Research Group laboratory (SUN-IRG), university of Stellenbosch, Cape Town, South Africa.The concentrations of 29 cytokines including pro-inflammatory cytokines , chemokines ; anti-cytokines (IL-10 and IL-1receptor antagonist: RA) and growth factors , were investigated in sputum samples from all study participants. Reagents were purchased from Merck Millipore, Billerica, Massachusetts, United States of America (USA). Experiments were performed and read on the Bio-Plex platform , with the Bio-Plex Manager Software version 6.1 used for bead acquisition and analysis.P-values ≤0.05 were considered significant.The data was analyzed using Graphpad Prism version 5 . Quantitative variables were presented as mean (± standard error) when the distribution was considered normal; if not they were represented by their median . A chi-square test was used to compare proportions. The Student’s t-test or Mann-Whitney U test was used to compare means or medians respectively between two groups. The ANOVA test was used to compare the quantitative variables between three groups at the same time. The Pearson’s chi squared test was used to test the association between the concentrations of cytokines and spirometric data.